Isomerase regulation of potassium channel trafficking and function. Kv4.2 channels are key determinants of dendritic excitability and integration, spike timing-dependent plasticity and long-term potentiation. Downregulation of Kv4.2 channel expression occurs following hippocampal seizures and in epilepsy suggesting A-type currents as targets for novel therapeutics. To identify Kv4.2 binding proteins, staff scientist Jiahua Hu employed a tandem affinity purification approach (TAP)to isolate the Kv4.2 protein complex from hippocampal neurons. Mass-spectrometry analysis identified known proteins such as KChIP family members and DPP6/10. The TAPMS assay also identified an isomerase as a binding partner of Kv4.2. The binding was confirmed by brain co-immunoprecipitation, co-expression in HEK293T cells, and peptide pull downin vitro. The isomerase binds to a specific Kv4.2 site, and the association is regulated by neuronal activity and seizure. To determine if and how the isomerase regulates the trafficking of Kv4.2, postbac Travis Tabor generated bungarotoxin binding site-tagged Kv4.2 at the second extracellular loop for visualizing Kv4.2 in live neurons. The bungarotoxin binding site-tagged Kv4.2 showed similar channel properties as WT Kv4.2 in biochemical and electrophysiological assays. The isomerizing activity may also regulate Kv4.2 binding to its auxiliary subunits. These data suggested that the isomeraseplays a role in regulating Kv4.2 function. To further study the physiological function of isomerase and Kv4.2 channel, we generated a knockin (KI) mousein whichthe isomerase binding site is specifically abolished using Crispr-Cas9 techniques. These mice are viable and appear normal. They showed normal initial learning and memory in Morris Water Maze. However, these Kv4.2 KI mice showed better reversal learning in Morris Water Maze than WT. In the operant reversal lever press, the KI mice displayed improved reversal learning. These data strongly support the idea that activity-dependent regulation of Kv4.2 plays an important role in cognitive flexibility. Cognitive flexibility is the ability to appropriately adjust ones behavior according to a changing environment. Cognitive flexibility is impaired in various neurodevelopmental disorders such as autism spectrum disorder (ASD). In light of these findings, postdoc Cole Malloy investigated how isomerization of Kv4.2 impacts neuronal function using whole-cell patch clamp electrophysiology in acute hippocampal slices. He utilized current-clamp recordings to detect alterations in action potential firing properties in the knock-in mice. Pharmacological manipulation of isomerase and kinase activity are underway to address the dependence of phosphorylation and/or conformation change induced by the isomerase to gain further insights into the molecular cascade impacting Kv4.2 function. Furthermore, given the behavioral results showing altered cognitive flexibility, experiments investigating synaptic function and plasticity in the KI mice are planned. Ca2+ regulation of potassium channel function. In addition to pore forming Kv4 subunits, native hippocampal A-type currents require non-conducting modulatory auxiliary subunits known as K-channel interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). Both KChIPs and DPLPs work in concert to enhance Kv4 function. Interestingly, in recent unpublished work we have identified a mechanism by which Kv4.2 current density is regulated by Ca2+ via R-type voltage gated Ca2+ channels (Cav2.3). Ca2+ regulation of Kv4.2 channels occurs despite an apparent lack of the structural determinants of the canonical Ca+-activated K+ channels. We hypothesized that KChIP auxiliary proteins imbue Kv4 channels with Ca2+ sensitivity as they contain four EF-hand domains, two of which bind Ca2+. To assess this possibility, postdoc Jon Murphy expressed a short KChIP isoform, KChIP2c with Kv4.2 in HEK293T cells and performed whole-cell patch clamp recordings in either nominal or 10 micromolar Ca2+. Under low Ca2+ conditions coexpression of KChIP2c enhances Kv4.2 function in several ways. KChIP2c increases K+ current density, shifts the voltage dependence of inactivation to more hyperpolarized potentials and accelerates recovery from inactivation. In the presence of 10 micromolar Ca2+, he measured a 1.5 fold increase in peak Kv4.2 current density while the other parameters of Kv4 function remain unchanged. Intriguingly, while the boosting effect of Ca2+ is conserved among the Kv4 family including Kv4.1-4.3, it was only observed for short splice isoforms of KChIP2, KChIP2b/c and the predominant KChIP3 isoform, KChIP3a. KChIP mRNAs contain multiple start sites that generate considerable N-terminal variation and functional diversity in shaping A-type currents. While the KChIP core is cytoplasmic by nature, the variable N-termini regulate subcellular localization by encoding variable membrane interaction motifs. Comparisons of the variable N-terminal domains of KChIP2 isoforms suggested that a conserved polybasic domain limits Ca2+ sensitivity of longer KChIP2 isoforms. Ongoing studies are aimed at determining whether this previously unidentified polybasic domain regulates the plasma membrane trafficking and Ca2+ sensitivity of Kv4-KChIP2 complexes. Proteomic and subcellular localization studies suggest, that Cav2.3-containing voltage gated calcium channels could be a potential calcium source for a modulatory effect on Kv4.2-mediated A-type K currents (IA) in CA1 hippocampal neurons. Postdoc Jakob Gutzmann compared wild type with Cav2.3 knock-out neurons, and saw a significant reduction in somatic IA. Cav2.3 KO neurons showed a higher frequency of action potential firing after somatic injection of positive current. Further investigation revealed that individual action potentials showed profound changes in waveform. Longer time to peak, and more significantly, a broader full-width at half-maximum. Additionally, the fast afterhyperpolarization following an action potential was shifted towards positive potentials in cells from the KO animals. Ultimately, he could track down these changes pharmacologically to a functional lack of large conductance potassium (BK) channel activity. This is the first time that Cav2.3 has been linked to BK channel activity, which so far has been thought to be regulated by other calcium channels. To support this novel finding, Lin Lin also performed co-immunoprecipitation experiments from acutely isolated adult mouse hippocampal tissue, and could successfully co-IP BK and Cav2.3, which hints at a physical interaction between the two channels, in addition to the functional interaction described above. A consequence of the altered action potential waveform in Cav2.3 KO neurons is an increase in short-term plasticity between CA1, and the informationally downstream lying subiculum pyramidal neurons. DPP6 plays a role in Brain Development, Function and Behavior DPP6 is well known as an auxiliary subunit of Kv4.2 which has been associated with numerous developmental and intellectual disorders and neuropsychiatric pathologies, especially ASD. We have reported previously, a novel role for DPP6 in regulating dendritic filopodia formation and stability, affecting synaptic development and function. This year we found DPP6 knockout mice are impaired in learning and memory. Results from the Morris water maze, T-maze, Objects spatial location, Novel Object Recognition and Cued and fear conditioning tasks showed that DPP6-KO mice exhibit slower learning and reduced memory performance.